| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 47, 46983-46993, November 21, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Pharmacology and Toxicology, Walther Oncology Center/Walther Cancer Institute and Indiana University Cancer Center, School of Medicine, Indiana University, Indianapolis, Indiana 46202
Received for publication, May 12, 2003 , and in revised form, August 29, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
and PDGFR-
) and regulate diverse cellular functions (1–3). PDGF in general has been implicated to play an important role in embryogenesis, wound healing, as well as in the development of several serious disorders, including certain malignancies, atherosclerosis, and various fibrotic conditions. Understanding the mechanism of regulation of PDGF expression is, thus, important for designing strategies to control the expression of PDGF in such disorders.
The regulation of human PDGF-B expression is complex. The human PDGF-B gene contains seven exons spanning 24 kb of the genomic DNA on human chromosome 22. Transcription is normally driven by a short basal TATA-containing promoter that is responsible for production of the regular 3.8-kb transcript (4–7). However, this mRNA contains a GC-rich 5'-untranslated region (5'-UTR) of 1022 bases with three AUG codons and a highly stable secondary structure. This type of 5'-UTR sequences normally have an inhibitory effect on translation. Indeed, it has been found that the high level of the 3.8-kb PDGF-B mRNA is not accompanied by detectable proteins in many cell lines (8–10). In contrast, the PDGF-B protein is detected in some cell lines despite the presence of low levels of the 3.8-kb mRNAs (11). An alternative 2.8-kb transcript that lacks the long 5'-UTR sequence was detected in a few tumor cell lines and was shown to be associated with high level of PDGF-B protein (12). The 2.8-kb mRNA was also reported in cultured human renal microvascular endothelial cells (HRMECs) upon stimulation with transforming growth factor (TGF-) or phorbol 12-myristate 13-acetate (13). Interestingly, the 2.8-kb mRNA is degraded in a protein synthesis-dependent pathway and can be selectively enriched by cycloheximide treatment. The 2.8-kb mRNAs have also been detected in rat brain at certain stages of brain development, and its detection was associated with the increased level of PDGF-B protein (12). Thus, it has been suggested that the expression of PDGF-B mRNA with short 5'-UTR sequences possibly exists widely in non-transformed tissues in vivo (12).
One of the major sites of PDGF synthesis is within bone marrow megakaryocytes, the platelets progenitor cells. Because human erythroleukemia K562 cells are differentiated into megakaryocytes upon TPA stimulation, regulation of PDGF-B expression in K562 cells has been extensively studied. The 3.8-kb species of PDGF-B mRNA was dramatically increased upon TPA-induced differentiation (14). Cis-elements and transcription factors, which are responsible for such induction, were also identified for the TATA-containing promoter (15, 16). Recently, the 5'-UTR of the 3.8-kb transcript has been reported to contain an internal ribosome entry site (IRES), which became more active in K562 cells upon differentiation into megakaryotic cells (17, 18). The IRES-mediated translation initiation works by directly recruiting the translational machinery to the nearby AUG translation start codon and therefore effectively avoids the inhibition of the long structured 5'-UTRs (reviewed in Refs. 19–21). However, whether this IRES really exists is questionable due to the inevitable technical problem associated with the traditional dicistronic test (22, 23). Like most of the previous cellular IRES studies, the 5'-UTR of PDGF-B is claimed to contain IRES based on transfection of artificial dicistronic plasmid followed by Northern blot analysis. Due to its low sensitivity, the use of Northern blot cannot differentiate IRES from cryptic promoter or potential splicing activities, all of which generate the same effect in dicistronic DNA test (25).
Herein, we report our surprising finding that the 2.8-kb human PDGF-B mRNA, previously detected in HRMECs (13), is also produced in K562 cells upon TPA-induced differentiation. This transcript represents a minor species in Northern blot analysis. However, treatment of cycloheximide stabilizes the 2.8-kb transcript and enables its clear detection by Northern blot, 5' rapid amplification of cDNA ends, and RNase protection assay. Using luciferase reporter promoter assay and Northern blot analysis, we demonstrated that the DNA sequence encoding the 5'-UTR of the long PDGF-B mRNA contains promoters that function in various cell lines and produces two mRNA species with a medium and a short 5'-UTR, respectively. However, in the presence of the upstream TATA-containing promoter, only the transcript with a short 5'-UTR was produced in addition to the transcript with the full-length 5'-UTR, which may explain why only the 3.8-kb and the 2.8-kb endogenous mRNAs are produced. On the other hand, the 5'-UTR sequence of the long PDGF-B transcript severely inhibits translation and does not display any IRES activity when tested using more stringent methods such as RNA transfection and promoterless dicistronic assay. Therefore, the 3.8-kb mRNA may contribute little, if any, to the production of PDGF-B protein. Based on the above observations, we conclude that the major TATA-containing promoter and the promoters in the 5'-UTR work together to control the expression of the 2.8-kb PDGF-B transcript in a variety of cells as an effective source of mRNA for protein production. Tight control of the production of the 2.8-kb mRNA and its stability may be used widely to control PDGF-B expression, both constitutively and upon stimulation.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-galactosidase was purchased from Tropix. The pTR-GAPDH-Human (pTR-glyceraldehyde-3-phosphate dehydrogenase-Human), the RNA Century Marker, the MAXI-script in vitro transcription kit, and RPA III ribonuclease protection assay kit were products of Ambion. Cycloheximide and TPA were from Sigma. The plasmid pSM1 (24) containing PDGF-B DNA insert was obtained from ATCC. The human IMAGE cDNA (ID 5174750 and GenBankTM accession number BC029822
[GenBank]
) containing the partial 5'-UTR and the entire coding region of PDGF-B were purchased from Open Biosystems. Construction of Plasmids—The plasmid pBLHHCAT was a gift from Dr. William E. Fahl (McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, WI). The plasmid contains a 5.5-kb PDGF-B DNA fragment (HindIII-HindIII), which includes the full-length 5'-UTR and the proximal promoter (5). The DNA sequence for the long 5'-UTR was amplified using the following primers: JB7, 5'-CCCCACTAGTGGCAACTTCTCCTCC and OS35, 5'-CCCCCCATGGCGACTCCGGGCCCGGCCC (18). The purified PCR product was cloned into pRF, and pRF(-P) vector (25), resulting in pR-PDGF-F and pR-PDGF-F(-P), respectively. To construct the plasmid containing the 5' region, -807 to +1022, of PDGF-B (numbered relative to the known transcription start site in the remaining text unless otherwise specified), pHHCAT was first digested with HpaI and XhoI to isolate a 1.2-kb fragment that includes -807 to +475, which was then used to replace the SpeI/XhoI fragment in pR-PDGF-F(-P). To obtain the pRF(-P)-based construct that contains -807 to +82 of PDGF-B 5' region, a NheI/AvrII fragment isolated from the -807 to + 1022 construct was cloned into pRF(-P) at the NheI/NcoI sites.
Mutagenesis of the TATA box was performed using the Stratagene QuikChange XL mutagenesis kit according to the manufacturer's protocol. The sense primer for mutagenesis was 5'-CGCACTCTCCCTTCTCCCTCTAGATGGCCGGAACAGCTGAAAG-3' (with mutations shown underlined and in boldface). This primer changed the palindrome TTTATAAA sequence (the overlapping TATA box in both sense and antisense strand) into CTCTAGAT sequence that bears an XbaI site but shows no known binding sites for any transcription factors.
The 5'-end deletions of the 5'-UTR were generated using PCR for constructs containing the sequence from +75, +150, +225, +300, +375, +525, +600, or +675, to +1022. The other constructs containing sequence from +475, +769, or +862, to +1022 were obtained by deleting the SpeI/XhoI, SpeI/BamHI, and SpeI/SmaI fragments from the pR-PDGF-F (-P), respectively. The 3'-end deletion constructs containing +1 to +300, +1 to +395, +1 to +475, and +1 to +769 were obtained by deleting the RsrII/NcoI, SmaI/NcoI, XhoI/NcoI, and BamHI/NcoI fragments from pR-PDGF-F(-P), respectively. The construct containing +1 to +675 region of the 5'-UTR was obtained using PCR.
Dicistronic constructs containing poly(A) for in vitro transcription (Fig. 4) were engineered using the vector pSP64 Poly(A), which has 30-bp dA:dT. The EcoRV-XbaI fragment of the pRF vector containing Renilla luciferase gene was first cloned into pSP64 Poly(A) vector at the XbaI and blunted HindIII sites to generate the plasmid pSP-RA30. The XbaI fragment of pR-HRV-F (25) containing the IRES of HRV and the firefly luciferase gene was then isolated and cloned into pSP-RA30 at the XbaI site to generate pSP-R-HRV-FA30. The pSP-RFA30 plasmid was obtained by removing the IRES sequence of HRV from the pSP-R-HRVFA30 by digestion with SpeI and NcoI. To engineer pSP-R-PDGF-FA30, the SpeI-NcoI fragment from pR-PDGF-F, which contains the +1 to +1022 sequence, was used to replace the HRV IRES fragment in pRHRV-FA30 construct.
|
In Vitro Transcription and Translation—In vitro transcription and translation were performed as previously described (25). The pSP64 poly(A)-based dicistronic and monocistronic plasmids were linearized by using EcoRI. The capped transcripts were synthesized using the Sp6 RNA polymerase transcription system in the presence of 1 mM m7GpppG. DNA templates were digested using DNase, and the in vitro transcripts were purified using a Qiagen RNeasy Mini kit and quantified. For in vitro translation, 25 ng of the capped RNA transcripts was used to program translation in rabbit reticulocyte lysate in a final volume of 10 µl. Translation products were measured for firefly luciferase activities.
Cell Culture, DNA, and RNA Transfection—HeLa cells were maintained in Dulbecco's modified Eagle's medium, whereas H1299 and K562 cells were maintained in RPMI 1640 media both supplemented with 10% fetal bovine serum at 37 °C with 5% CO2.
DNA transfection in both HeLa and H1299 cells were performed with LipofectAMINE plus reagents according to the manufacture's protocol. In a 24-well plate, 
1 x 105 cells/well were plated and transfected with 0.4 µg of DNA. Cells were harvested 24 h following transfection for luciferase assay.
RNA transfection was performed using the cationic liposome-mediated method as previously described (25). Briefly, 2 x 105 cells/well were seeded onto 6-well plates on the day before transfection. Cells were washed once with Opti-MEM I-reduced serum medium (Invitrogen) and left in the incubator with some medium during preparation of the liposome-polynucleotide complexes. One milliliter of Opti-MEM I medium in a 12 x 75-mm polystyrene snap-cap tube was mixed with 12.5 µg of Lipofectin reagent and 5 µg of capped mRNA. The liposome/RNA/medium mixture was immediately added to cells. Eight hours following transfection, cells were harvested and processed for luciferase analysis.
Transient Transfection of K562 Cells—Transient transfections of K562 cells were performed using electroporation. K562 cells were collected by centrifugation, washed with phosphate-buffered saline, and resuspended in the same buffer at 107 cells per 0.4 ml. For each electroporation, 0.4 ml of cell suspension was mixed with 20 µg of constructs and 1.0 µg of pCMV-gal in a final volume of 0.5 ml. Each electroporation pool received an electric pulse of 240 V and 1025 microfarads. After electroporation, the cells were incubated in 20 ml of prewarmed medium containing 20% serum for 24 h. The cells were then divided into two dishes with one supplemented with TPA (2 ng/ml) and other the same volume of solvent (ethanol) as a control. Forty-eight hours after TPA addition, the cells were collected by centrifugation, washed with phosphate-buffered saline, and lysed in passive lysis buffer and dual-luciferase, and -galactosidase activity were determined.
Northern Blot Analysis—Subconfluent H1299 cells in 10-cm plates were transfected with 4 µg/plate constructs using LipofectAMINE Plus. Twenty-four hours following transfection, the total RNAs were extracted using an RNeasy Mini kit and digested with RNase-free DNase to remove residual plasmid DNA. The poly(A) RNAs were then isolated from 250 µg of total RNAs using an Oligotex mRNA Mini kit. One-fifth of the mRNAs were separated in 1% agarose gels in the presence of formaldehyde and MOPS buffer and blotted onto MAGNA nylon membranes. The blots were then hybridized with a 32P-labeled firefly luciferase DNA probe (1656 bp), which was isolated by cleaving pRF with NcoI and XbaI and labeled using the Rediprime II random prime labeling system. The glyceraldehyde-3-phosphate dehydrogenase mRNA was also detected using specific probes as a control.
For Northern blot analysis of PDGF-B expression in K562 cells, the total RNA was prepared from normal and differentiated K562 cells. The differentiation of K562 cells into megakaryocytes was induced by TPA (2 ng/ml) treatment for 2 days. Cycloheximide (10 µg/ml) was added to the medium on the third day following TPA treatment to stabilize the 2.8-kb mRNA. The poly(A) mRNA was isolated, and 2 µg of mRNA of each sample was used for Northern blot analysis as described above. The probe used for Northern blot was a 2-kb fragment isolated from pSM1 (24) by BamHI digestion.
PCR Analysis—Determination of the 5'-end of the 2.8-kb species was performed with the 5' RACE system for rapid amplification of cDNA ends using the method provided by the supplier. Briefly, the first cDNA strand was synthesized using 5 µg of total RNA isolated from K562 cells that was cultured with TPA for 2 days and subsequently cultured with both TPA and cycloheximide for another 16 h. The primer used for cDNA synthesis is human PDGF-B-specific antisense primer, 5'-CCACTGTCTCACACTTG-3', located at 548 bases downstream of the translation start codon. The primers used for first-round PCR were 5' RACE Abridged Anchor Primer from the supplier (36 bases) and human PDGF-B-specific antisense primer with a sequence of 5'-GCTTCTTCCGCACAAT-3', located 488 bases downstream of the translation start codon. The primers used for the final round PCR were Abridged Universal Amplification Primer from the supplier and PDGF-B-specific primer, 5'-CTGACCAGACGCAGGTA-3', located at 56 bases downstream of the translation start codon. Takara Taq polymerase and GC buffer II were used for PCR amplification. The reaction conditions were 94 °C for 2 min followed by 40 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min. The PCR products were then purified, blunted with Klenow, and then cloned using the PCR Zero Blunt Cloning kit. Individual clones were isolated and sequenced to determine the transcription start sites.
Determination of the 5'-end of the longer transcript from the pRPDGF-F(-P) transcription was performed by RT-PCR. About 5 µg of total RNA isolated from pR-PDGF-F and pR-PDGF-F(-P)-transfected H1299 cells was used. The first strand of cDNA was synthesized using an antisense primer targeting firefly luciferase mRNA located 78 bases downstream of the translation start codon. PCR amplification was performed using antisense primer located at +675 and three sense primers located at +150, +225, +300, and +375 of the 5'-UTR of PDGF-B, respectively.
Ribonuclease Protection Assay—RPA was performed using the RPAIII kit according to the supplier's instructions. Briefly, the RNA probe was produced by first cloning into pGEM-4Z at the EcoRI and HindIII site the region -118 to +241 (numbered relative to the translation start site) of PDGF-B derived from the Image 5174750 (National Institutes of Health) cDNA clone by PCR. The resulting plasmid was linearized with EcoRI and transcribed using T7 RNA polymerase in the presence of 0.5 mM each of ATP, GTP, UTP, and 0.01 mM CTP supplemented with 3.12 µM [-32P]CTP. The 32P-labeled probe was digested with DNase and purified using a Sephadex G-25 Quick Spin column. About 0.5 x 106 cpm of the probe was hybridized to 2 µg of mRNA at 45 °C overnight followed by digestion with RNase T1/A at 37 °C. The reaction was then stopped, and the protected RNAs were precipitated before separation by electrophoresis on a 6% acrylamide/8 M urea gel for autoradiography.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
or phorbol 12-myristate 13-acetate stimulation. It was thought that the 2.8-kb mRNA was degraded through a protein synthesis-dependent pathway, because treatment with cycloheximide selectively stabilized the 2.8-kb mRNA but not the 3.8-kb mRNA. Fen and Daniel also demonstrated that the 2.8-kb mRNA did not arise from post-transcriptional processing of the 3.8-kb mRNA but rather possibly from internal promoter within the 5'-UTR. The cycloheximide treatment did not enhance transcription of PDGF-B but rather increase the half-life of the 2.8-kb mRNA. The 5'-truncated PDGF-B mRNA was also observed in a few tumor cell lines and increased in rat brain at a certain stage of brain development, and the level of the 5'-truncated mRNA was shown to be associated with PDGF-B protein level (12). We hypothesize that the 2.8-kb mRNA of PDGF-B may also be produced in K562 cells upon TPA-induced differentiation. To test this hypothesis, we first performed Northern blot analysis of the endogenous PDGF-B transcript in K562 cells and demonstrated that a major transcript of 3.8 kb was detected when K562 cells were treated with TPA for 2–3 days (Fig. 1A, lane 3). A minor transcript of 2.8 kb was also evident (lane 3) and more pronounced with longer exposure (data not shown). When cells were treated with cycloheximide for 6 or 16 h following TPA treatment, the 2.8-kb mRNA increased significantly (Fig. 1A, lanes 4 and 5). Treatment of K562 cells with cycloheximide alone, however, did not enhance expression of either mRNA species (Fig. 1A, lane 2), suggesting that cycloheximide alone did not affect PDGF-B transcription. Therefore, the TPA-stimulated K562 cells displayed a similar profile of PDGF-B transcription as HRMECs.
|
Because the results in 5'-RACE analysis could also be due to premature termination of polymerase reaction by the secondary structures in the 5'-UTR sequence, we performed a ribonuclease protection assay (RPA) using a PDGF-B probe spanning -118 and +241 (numbered relative to the translation start codon) (Fig. 1C). As shown in Fig. 1D, two fragments were found to be protected, and their sizes were estimated to be 289 and 212 bases, respectively (lane 3). While the longer protected fragment is possibly derived from the 3.8-kb transcript, the smaller fragment with 77 bases less is likely derived from the 2.8-kb transcript with shorter 5'-UTRs. It is noteworthy that the smaller fragment represents a significant species of protected fragments in samples treated either without (lane 3) or with cycloheximide (lane 4), suggesting the 2.8-kb transcript appears to be more efficiently protected. It also appears that the 2.8-kb transcript (the smaller fragment) is relatively more abundant upon cycloheximide treatment (compare lanes 3 and 4). Thus, these findings support the conclusion derived from the results shown in Fig. 1 (A and B) and confirm that the cycloheximide treatment selectively stabilizes the 2.8-kb mRNA.
The Long 5'-UTR Sequence of PDGF-B Inhibits Cap-dependent Translation—Although the 2.8-kb mRNA represents a minor species in TPA-induced PDGF-B transcripts, it may serve as the primary template for PDGF-B protein production, because the full-length PDGF-B transcript may not be efficiently translated due to translation inhibition by the long 5'-UTR. To test this concept, we determined the effect of the long 5'-UTR sequence of PDGF-B on translation. For this purpose, we constructed a series of plasmids that were used for generating in vitro transcripts with different lengths of 5'-UTRs upstream of a firefly luciferase reporter gene. These deletion mutants contain putative secondary structures with different free energies (
G) ranging from -421 to -15 kcal/mol as predicated by Zuker's mfold program (26) (Fig. 2A). The G value is an indicator of the stability of the secondary structure that may inhibit a ribosome-scanning process for cap-dependent translation (27). The higher the G value (with a minus sign), the more stable is the secondary structure. Capped in vitro transcripts were then generated (Fig. 2B), and equal amounts of the transcripts were used to program cell-free translation in rabbit reticulocyte lysate. The products were measured as firefly luciferase activity. As shown in Fig. 2B, the presence of the 5'-UTR with full-length (+1 to +1022) or 647 bases (+375 to +1022) severely inhibited the luciferase production. The transcript with a 5'-UTR of 253 bases (+769 to +1022) or 160 bases (+862 to +1022) is about 20-fold more efficiently translated than the one with a full-length 5'-UTR. Deletion of most of the 5'-UTR sequence (+983 to +1022) increased the translation by about 80-fold. These results suggest that the long 5'-UTR sequence of PDGF-B in the 3.8-kb transcript inhibits the translation of its downstream sequence, and the transcripts with short 5'-UTRs are much better templates for translation of a heterologous gene, consistent with previous findings (4, 28).
|
17-fold as compared with the vector control. In cells transfected with the promoterless vector pRF(-P), both Renilla and firefly luciferase activities were low. However, pR-PDGF-F(-P)-transfected H1299 cells displayed similar firefly luciferase activity as pRPDGF-F transfected H1299 cells, although there was no increase in Renilla luciferase activity. Similar results were also observed in experiments performed with HeLa cells (data not shown). These results strongly suggest that the long 5'-UTR sequence of PDGF-B contains promoter activity in both HeLa and H1299 cells.
|
The 5'-UTR Promoter Has Two Transcription Start Sites for Production of mRNAs with Shorter 5'-UTRs—To further determine whether the long 5'-UTR sequence of PDGF-B contains putative promoters, we tested whether there are any transcripts that are derived from the 5'-UTR sequence in cells transfected with the dicistronic constructs. For this purpose, poly(A) RNAs were isolated for Northern blot analysis from cells transfected with pRF, pR-PDGF-F, pR-PDGF-F(-P), and pRF(-R). The pRF(-R) construct, which lacks the Renilla luciferase gene, was used as a monocistronic control (25). It is expected to use the SV40 promoter to produce a transcript with a 5'-UTR of 100 bases and the firefly luciferase-encoding sequence. The pRF vector was used as a dicistronic control, which produces only a dicistronic mRNA using the SV40 promoter. As shown in Fig. 5A, a dicistronic transcript from control pRF (lane 2, indicated by an asterisk) and a monocistronic transcript from control pRF(-R) (lane 3, indicated by an arrowhead) were detected as expected. The dicistronic transcript derived from pR-PDGF-F (lane 1, indicated by an asterisk) has a slower mobility than the one from pRF (lane 2), consistent with the presence of the 5'-UTR sequence of PDGF-B in the intergenic region. Two additional transcripts were generated from pR-PDGF-F (lane 1, indicated by A and B), which have similar sizes to the monocistronic transcript derived from pRF(-R) (lane 3), suggesting that they may be monocistronic mRNA transcribed from the 5'-UTR sequence of PDGF-B located in the intergenic region. This conclusion was further confirmed by the production of the same two transcripts from pR-PDGF-F(-P) construct (lane 4), which lacks the vector SV40 promoter and thus the dicistronic product (lane 4). The bigger transcript (labeled as A) may have a longer 5'-UTR than the smaller one (labeled as B). The 5'-UTR sequence of the transcript B may be very short, because it was smaller than the monocistronic mRNA transcript generated from pRF(-R) (compare lanes 4 and 3). Based on the above observations, we conclude that the 5'-UTR sequence of PDGF-B may contain two discrete promoters (designated as P1 and P2) that mediate the production of two types of transcripts with medium and short 5'-UTR sequences, respectively. The transcripts with the short 5'-UTR is likely the one that can be much more efficiently translated than the one with medium and the original long 5'-UTRs as shown in Fig. 2.
|
To further delineate the boundaries of the DNA region that are responsible for promoter activities, systematic deletion mutants were created from either 5'- or 3'-end of the 5'-UTR of PDGF-B (Fig. 6A). These 5'-UTRs with deletions were engineered into the promoterless dicistronic vector and used to determine their ability to direct firefly luciferase expression in both H1299 and HeLa cells (Fig. 6B). It should be noted, however, that the results of the luciferase reporter assay may be subject to both transcriptional and translational control when mRNAs with different lengths of 5'-UTRs are produced (27). Thus, the data in Fig. 6 should be analyzed together with the Northern blot and RT-PCR result shown in Fig. 5B. The data in Fig. 6 clearly demonstrated that two discrete regions, i.e. the +1 to +395 (P1) and the +769 to +1022 (P2) sequence, contain promoter activity. This result confirmed the data in Fig. 5 where we detected two discrete transcripts (Fig. 5A, labeled as A and B in lane 4). The P1 promoter activity is about 3- to 4-fold of that of the P2 promoter. Because transcripts from the P1 promoter have a relatively long 5'-UTR, they can not be efficiently translated (see Fig. 2). The +1 to +769 construct does not contain the P2 sequence. However, because it contains the full P1 promoter, the +1 to +769 construct can produce a transcript with a shorter 5'-UTR that lacks the downstream major GC-rich region. As shown in Fig. 2A, the computed free energy for the +769 to +1022 is about -119 kcal/mol. Removal of this region is thus expected to result in an enhanced translation efficiency and, consequently, an increase in luciferase activity (Fig. 6, B and C). Further deletion from the 3'-end up to 675 bases resulted in further increase in luciferase activity (+675 to +1022). The luciferase activity started to drop in the +1 to +303 construct in both cell lines, indicating that the +303 to +395 region may contain enhancing elements for P1 promoter.
|
Function of the 5'-UTR Promoter in the Presence of the Upstream TATA-containing Promoter (P0)—The above promoter analysis focused on the 5'-UTR sequences only. However, the promoter activity of the 5'-UTR sequence may be different in the presence of the upstream TATA-containing P0 promoter. To address this issue, we generated a series of constructs as shown in Fig. 7A and analyzed the promoter activity using both luciferase assay (Fig. 7B) and Northern blot (Fig. 7C). The longest construct, from -807 to +1022, contained both the TATA-containing promoter (P0) and the full-length 5'-UTR sequence. It has been known that the TATA-box is critical for constitutive promoter activity for P0 promoter and, thus, a TATA-box mutant construct (from -807 to +1022 mTATA with mutation from TTTATAAA to CTCTAGAT, which has no homology with any known transcription factor binding sites) was also generated to determine the effect of P0 promoter on expression from the -807 to +1022 sequence. Two other constructs (those from -807 to +87 and -807 to +87 mTATA) were also generated to determine the P0 activity in the absence of the 5'-UTR. These plasmids were transfected into HeLa, H1299, and HEK293 cells for promoter analysis. As shown in Fig. 7B, the -807 to +1022 construct has about a 3-, 4-, and 2-fold increase in luciferase expression in HeLa, H1299, and HEK293 cells, respectively, as compared with the control +1 to +1022 construct (pR-PDGF-F(-P)) that has only the 5'-UTR sequence. Mutation of the TATA-box almost eliminated the enhancement, suggesting that the integrity of the P0 promoter affects transcription from the -807 to +1022 construct. On the other hand, removal of the 5'-UTR sequence (-807 to +87 construct) enhanced the reporter expression about 4- to 5-fold compared with the full-length construct (-807 to +1022), indicating that the 5'-UTR caused an inhibitory effect on luciferase expression in the -807 to +1022 construct. This observation is consistent with the data shown in Fig. 2B. The -807 to +87 mTATA construct had about 20–30% of luciferase activity compared with the -807 to +87 construct, which again demonstrate that the TATA-box is critical for the activity of the P0 promoter.
|
Induction of the P0, P1, and P2 Promoter during Megakaryocytic Differentiation—We next examined the induced activity of the 5'-UTR promoter and the major P0 promoter in K562 cells upon TPA stimulation. For this purpose, K562 cells were transiently transfected with various constructs (Fig. 8A) by electroporation and were then treated without (Fig. 8B) or with (Fig. 8C) TPA for 2 days followed by measuring luciferase activities in cell lysates. The vector control was used to normalize the luciferase activities. As shown in Fig. 8B, the 5'-UTR sequence (+1 to +1022) alone stimulated about 10-fold expression compared with the vector control in K562 cells. In the presence of both P0 promoter and the 5'-UTR sequence (-807 to +1022), the activity increased another 2.6-fold. However, mutation of the P0 promoter eliminated this increase (-807 to +1022 mTATA). Deletion of the 5'-UTR sequence dramatically increased the expression of luciferase in K562 cells (11-fold increase) (compare the -807 to +82 range with that from -807 to +1022), suggesting that the long 5'-UTR sequence inhibits translation. These observations are consistent with the results derived from 293, HeLa, and H1299 cells shown in Fig. 7.
|
2.7-fold (Fig. 8C). Interestingly, this result is in agreement with a 2–3-fold increase of "IRES" activity by induction observed previously (29), suggesting that the promoter activity of the 5'-UTR sequence might be misinterpreted as IRES activity. In the presence of the P0 promoter, however, the production of the luciferase reporter increased 10-fold by TPA stimulation (compare -807 to +1022 with +1 to +1022). The induction of luciferase expression of P0 promoter without the 5'-UTR sequence was 20-fold (-807 to +87), demonstrating that the P0 promoter is the major promoter that responds to TPA stimulation as compared with the 5'-UTR promoter. The luciferase activity from the construct containing P0 and 5'-UTR (the P1 and P2 promoter) increased about 10-fold, which is higher than the increase observed with the 5'-UTR promoter (P1 and P2) alone (2.7-fold) but lower than that by the P0 promoter alone (20-fold). Disruption of the integrity of the upstream promoter by mutation of the TATA box significantly decreased the production of luciferase protein from 20- to 5-fold (Fig. 8C). However, the -fold increase in luciferase activity with the construct containing mutated TATA box was still higher than that with the 5'-UTR sequence alone. This is consistent with results previously reported by others (6, 16) that ciselements other than TATA signals in the P0 promoter also contribute to TPA induction. Because the transcript derived from the P0 promoter contains the full-length 5'-UTR that inhibits translation and only the transcripts derived from P2 can be efficiently translated, we propose that the transcription from the 5'-UTR promoter is greatly enhanced in the presence of the upstream promoter by TPA stimulation.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
By using RNA transfection, we demonstrated that dicistronic mRNAs bearing a full-length 5'-UTR of PDGF-B in the intergenic region does not enhance the translation of the downstream cistron (Fig. 4). Although in a conventional dicistronic DNA transfection assays, cells transfected with a dicistronic construct, which contains the full-length 5'-UTR of PDGF-B in the intergenic region, showed an enhanced expression of the second cistron, no significant decrease in the expression of the second cistron was observed after the SV40 promoter of the vector was removed (Fig. 3). These observations suggest that the 5'-UTR sequence of PDGF-B contains promoters that underlie the enhanced expression of the second cistron observed in the traditional dicistronic DNA assay. We also found that the 5'-UTR promoter activity increased by about 2- to 3-fold in K562 cells undergoing megakaryocytic differentiation following TPA treatment (Fig. 8). This increase is surprisingly similar to the reported differentiation-induced IRES activity (18, 29). Thus, it is likely that the previously claimed IRES activity of PDGF-B was misinterpreted from the promoter activity present in the 5'-UTR sequence of PDGF-B. In light of these findings, we propose that the effective production of PDGF-B protein mainly relies on the production of the transcripts with shorter 5'-UTRs generated from transcription using the 5'-UTR promoter.
Although the existence of promoter activities in the 5'-UTR sequence of PDGF-B has been proposed previously (10, 12, 13), we, for the first time, characterized the 5'-UTR promoter and demonstrated that they are inducible and may be responsible for the previously thought IRES activity. The 5'-UTR promoter activity showed about 5–10% of the activity of the major TATA-box containing P0 promoter in various cell lines tested when analyzed by the luciferase reporter promoter assay (Fig. 7B). As shown by Northern blot analysis, the 5'-UTR promoter of PDGF-B can by itself initiate transcription from discrete locations, with one located around 800 bases upstream (Fig. 5B), and the other is very close to the translation start codon (Figs. 5 and 7). Detailed promoter analysis revealed two distinctive promoter regions, located approximately in position +150 to +303 (P1) and +769 to +1022 (P2) of the 5'-UTR sequence, respectively (Fig. 6). The P1 promoter in the 5'-UTR is about 3- to 4-fold more active than P2 in both HeLa and H1299 cells (Fig. 6).
We further investigated the function of the 5'-UTR promoter in the presence of the major TATA-box containing promoter (P0) (Fig. 7). TATA-box has previously been shown to be critical for proper function of the P0 promoter activity (5). This is confirmed in our promoter assay using both reporter luciferase activity assay and Northern blot analysis (compare the result of the -807 to +87 construct with that of the -807 to +87 mTATA construct). Although the major promoter P0 alone is about 7- to 20-fold more active than the 5'-UTR promoter (compare the result of the -807 to +87 construct with that of the +1 to +1022 construct), the luciferase activity increased only 2- to 4-fold when they coexist. Mutation of the TATA-box in the -807 to +1022 construct caused the luciferase activity to drop to the similar level to that of the +1 to +1022 construct. Although the P2 promoter functions in all of the constructs, the P1 promoter functions only in the absence of the P0 promoter or in the presence of the P0 promoter with a mutated TATA box (see Fig. 7C). Furthermore, the transcript generated from the P0 promoter with the -807 to +1022 mTATA construct did not cause any increase in the luciferase activity produced. Thus, it is likely that the P0 and P1 promoter are integrated into one promoter and drives transcription of the 3.8-kb full-length transcript that can be poorly translated in vivo.
We also analyzed the induction of the promoter activities in K562 cells upon TPA-induced megakaryocytic differentiation (Fig. 8) and demonstrated that the P0 promoter is a major player to respond to TPA stimulation, whereas the P1 and P2 promoters in the absence of P0 are induced only in a minor fashion. However, when P0 and the 5'-UTR promoter coexist, the overall protein production increased about 10-fold upon induction in transient transfection assays. Stable clones transfected with the -807 to +1022 construct with all three promoters displayed a similar induction profile.2 Based on these observations, we propose that transcriptions from the 5'-UTR promoters are greatly induced by TPA in the presence of the P0 promoter, and P0 and the 5'-UTR promoters work in a highly coordinated manner to tightly control the production of transcripts both with the long and short 5'-UTRs and, therefore, the constitutive and induced production of PDGF-B protein. Although cis-elements and transcription factors has been well characterized for the P0 promoter (5, 7, 15, 16, 30, 31), further studies is clearly needed to characterize the 5'-UTR promoter. Nevertheless, our experiments using Drosophila SL2 cells transfected with 5'-UTR promoter constructs demonstrated that Sp1 might be one of the transcription factors that can trans-activate the 5'-UTR promoter.2
Our promoter analysis strongly supports the argument that the 2.8-kb PDGF-B mRNA with short 5'-UTR is likely derived from transcription from the P2 promoter of the 5'-UTR sequence as previously suggested (10, 12, 13), although there is no evidence available for the function of the natural P2 promoter of PDGF-B on human chromosome 22. The integrated P0/P1 promoter likely drives the production of the 3.8-kb full-length PDGF-B mRNAs. The finding that the dramatic increase of 2.8-kb mRNA species in K562 cells following TPA treatment (Fig. 1A) contradicts with only a 2-fold induction of the 5'-UTR promoter activity in the absence of P0 (Fig. 8C) and argues for a coordination between the P0/P1 promoter and the P2 promoter.
In the analysis of PDGF-B mRNA in K562 cells upon TPA-induced differentiation, we clearly detected a 2.8-kb mRNA that has a 5'-UTR of about 15–27 bases. The 2.8-kb mRNA was not reported for K562 cells in previous studies, possibly because it represents only a minor species and is not evident in Northern blot with short exposures. We, however, noticed that a Northern blot performed by Colamonici et al. (14) clearly showed a 2.8-kb PDGF-B mRNA in K562 cells that were treated for 3 or 4 days (see Fig. 2 in Ref. 14), although it is not discussed in this report. Similarly, Fen and Daniel (13) reported the detection of a 2.8-kb mRNA species in human renal microvascular endothelial cells (HRMECs) upon treatment with either TGF- or TPA. The 5'-truncated mRNAs were also detected in a few tumor cells and in rat brain tissue at a certain stage of development, and its level correlates the level of PDGF-B protein (12). Based on these observations, we propose that the production of the 2.8-kb mRNA may be tightly regulated and widely used for effective protein production of PDGF-B, both constitutively and upon induction by biological stimuli. However, it remains unknown what is the role of the 3.8-kb mRNA of PDGF-B if it cannot be used as template for efficient protein synthesis.
Consistent with Fen and Daniel (13), the 2.8-kb mRNA in TPA-induced K562 cells is selectively enriched by cycloheximide treatment. Although cycloheximide likely does not affect the transcription of PDGF-B (see Fig. 1, lanes 1 and 2), we cannot rule out the possibility that cycloheximide treatment may inhibit the synthesis of a suppressor for transcription and thus, results in the increase in the 2.8-kb mRNA. However, the analysis of PDGF-B mRNA half-life by Fen and Daniel demonstrated that the 2.8-kb mRNA undergoes rapid decay without cycloheximide treatment, arguing for that cycloheximide treatment is associated with decay of the 2.8-kb mRNA. On the other hand, the 2.8-kb mRNA is more abundant in some tumor cells and in developing rat brain tissues at a certain stage (10, 12). Such abundance may likely be a result of an increase in either transcription from the P2 promoter or alternatively an increase in mRNA stability. The regulation of mRNA stability has been demonstrated to play a major role in regulating the expression level of oncogene and cytokine mRNAs during cell growth, differentiation, and neoplastic transformation (32). The mRNA decay rates are regulated by cis-acting sequence determinants, mRNA-binding proteins, endo- and exo-ribonucleases, and translation (32). For example, c-myc encoding region contains a coding region instability determinant (CRD), located in the last 249 nucleotides of the coding region, which mediates rapid turnover of c-myc mRNA (33). The CRD-mediated turnover is translation-dependent and blocked by translation inhibitor cycloheximide. The CRD-BP protein specifically binds to the CRD region and prevented c-myc mRNA from degradation (34). CRD-BP expression parallels c-myc expression during liver development. Interestingly, the abundance of the 2.8-kb mRNA of PDGF-B is also developmentally regulated in rat brain (12). Because the level of the 2.8-kb mRNA species is a major determinant of PDGF-B protein level (10, 12), alteration of the stability may be another level of control in PDGF-B expression regulation. How the stability of the 2.8-kb mRNA is regulated remains to be an intriguing question for further studies.
|
FOOTNOTES |
|---|
To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, IUCC, Indiana University School of Medicine, 1044 W. Walnut St., R4-166, Indianapolis, IN 46202. Tel.: 317-278-4503; Fax: 317-274-8046; E-mail: jianzhan{at}iupui.edu.
1 The abbreviations used are: PDGF, platelet-derived growth factor; SL2, Schneider's Drosophila cell line 2; 5'-UTR, 5'-untranslated region; IRES, internal ribosome entry site; HRV, human rhinovirus; LUC, luciferase; TPA, 12-O-tetradecanoylphorbol-13-acetate; HRMEC, human renal microvascular endothelial cell; TGF-, transforming growth factor ; RACE, rapid amplification of cDNA ends; CMV, cytomegalovirus; Mops, 4-morpholinepropanesulfonic acid; RPA, ribonuclease protection assay; CRD, coding region instability determinant.
2 B. Han, Z. Dong, and J.-T. Zhang, unpublished observation.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
